Many proteins are glycosylated, that is, they are conjugated to carbohydrates, forming a conjugate known as a glycoprotein. Often, the carbohydrates are oligomers that consist of several monosaccharide residues, which form a oligosaccharide, or glycan. These polymers can form straight or branched chains. When the carbohydrate is conjugated to the protein through a nitrogen atom, typically of an asparagine residue, the carbohydrate is referred to as an N-glycan.
Glycosylation is a post-translational modification of the protein, so it occurs after the protein is translated from its encoding nucleic acid. Since the carbohydrates conjugated to a protein can affect pharmacokinetics, stability, bioactivity or immunogenicity, determining the composition of the carbohydrates attached to a protein is important. Characterization of carbohydrates is required for biologics (therapeutic glycoproteins, vaccines, etc.) to show composition of matter and consistency of manufacture, resulting in a need for extensive characterization of product. Thus, the ability to remove some or all of the glycans from a protein and to analyze the carbohydrate or the protein, or both, to determine their composition or compositions is useful both for quality control and often for determining whether a protein will have a desired therapeutic efficacy or other effect.
For most analytical techniques, the glycans must be released or removed from the protein before analysis can be performed. Glycan removal, or “deglycosylation”, is achieved by one of two methods, enzymatic digestion or chemical release. In enzymatic digestion, for example, N-glycans are released from glycoproteins by enzymatic cleavage with PNGase F (Peptide-N4-(acetyl-β-glucosaminyl)-asparagine amidase, EC 3.5.1.52) or other endoglycosidases such as endo-alpha-N-acetyl-galactosaminidase, Endoglycosidase F1, Endoglycosidase F2, Endoglycosidase F3, Endoglycosidase H, and the like, and are then treated to label their free-reducing terminus with a fluorescent dye for analysis by methods such as high performance liquid chromatography (HPLC), capillary electrophoresis (CE), carbohydrate gel electrophoresis, or mass spectrometry. In chemical release, the glycoprotein is typically subjected to hydrazinolysis or trifluoromethanesulfonic acid (TFMS). TFMS, for example, removes all N- and O-linked glycans. Unfortunately, it also destroys the carbohydrates, rendering it unsuitable for use in applications in which analysis of the carbohydrates is important. Both the enzymatic and the chemical deglycosylation procedures encompass multiple steps, extended incubation times, and clean-up steps prior to analysis of the released glycans.
Fluorescent labeling of glycans facilitates their sensitive detection as well as contributes to improved resolution. Released N-glycans are most commonly labeled by reductive amination, where the free-reducing end of a glycan is conjugated to the free amino group of a fluorescent dye. Fluorescent labeling of glycans by reductive amination usually requires anhydrous conditions, elevated temperatures and extended incubation times, which may result in a partial degradation of labile constituents of N-glycans, such as sialic acids.
Therefore, there is a need for more rapid methods that can be performed under relatively mild conditions, that reduce handling and clean up steps, that are suitable for use with multiple analytic methods, and that improve the amount and types of glycans released. Surprisingly, the present invention meets these and other needs.